GH10 xylanase, GH62 arabinofuranosidase, milling process and other application

ABSTRACT

The present invention provides an improved process of treating crop kernels to provide a starch product of high quality suitable for conversion of starch into mono- and oligosaccharides, ethanol, sweeteners. The present invention also provides polypeptides having GH10 xylanase activity, polypeptides having GH62 arabinofuranosidase activity and the uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofinternational application no. PCT/CN2017/112865 filed Nov. 24, 2017,which claims priority or the benefit under 35 U.S.C. 119 ofinternational application no. PCT/CN2016/107281 filed Nov. 25, 2016. Thecontent of each application is fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

Field of the Invention

The present invention relates to an improved process of treating cropkernels to provide a starch product of high quality suitable forconversion of starch into mono- and oligosaccharides, ethanol,sweeteners, etc. The present invention relates to polypeptides havingGH10 xylanase activity and polypeptides having GH62 arabinofuranosidaseactivity. The present invention also relates to a process for extractionor separation of crude palm oil. Further, the invention also relates toan enzyme composition comprising one or more enzyme activities suitablefor the process of the invention.

BACKGROUND OF THE INVENTION Description of the Related Art

Wet milling is often used for separating corn kernels into its fourbasic components: starch, germ, fiber and protein.

Typically, wet milling processes comprise four basic steps. First thekernels are soaked or steeped for about 30 minutes to about 48 hours tobegin breaking the starch and protein bonds. The next step in theprocess involves a coarse grind to break the pericarp and separate thegerm from the rest of the kernel. The remaining slurry consisting offiber, starch and protein is finely ground and screened to separate thefiber from the starch and protein. The starch is separated from theremaining slurry in hydrocyclones. The starch then can be converted tosyrup or alcohol, or dried and sold as corn starch or chemically orphysically modified to produce modified corn starch.

The use of enzymes has been suggested for the steeping step of wetmilling processes. The commercial enzyme product Steepzyme® (availablefrom Novozymes A/S) has been shown suitable for the first step in wetmilling processes, i.e., the steeping step where corn kernels are soakedin water.

More recently, “enzymatic milling”, a modified wet-milling process thatuses proteases to significantly reduce the total processing time duringcorn wet milling and eliminates the need for sulfur dioxide as aprocessing agent, has been developed. Johnston et al., Cereal Chem, 81,p. 626-632 (2004).

U.S. Pat. No. 6,566,125 discloses a method for obtaining starch frommaize involving soaking maize kernels in water to produce soaked maizekernels, grinding the soaked maize kernels to produce a ground maizeslurry, and incubating the ground maize slurry with enzyme (e.g.,protease).

U.S. Pat. No. 5,066,218 discloses a method of milling grain, especiallycorn, comprising cleaning the grain, steeping the grain in water tosoften it, and then milling the grain with a cellulase enzyme.

WO 2002/000731 discloses a process of treating crop kernels, comprisingsoaking the kernels in water for 1-12 hours, wet milling the soakedkernels and treating the kernels with one or more enzymes including anacidic protease.

WO 2002/000911 discloses a process of starch gluten separation,comprising subjecting mill starch to an acidic protease.

WO 2002/002644 discloses a process of washing a starch slurry obtainedfrom the starch gluten separation step of a milling process, comprisingwashing the starch slurry with an aqueous solution comprising aneffective amount of acidic protease.

Palm oil is an edible vegetable oil which is obtained from the mesocarpof palm fruits. Palm fruits or fruitlets grow in large bunches. The palmfruitlets are stripped from the fruit bunches after being sterilized.The high temperature causes the enzymes naturally occurring enzymes inthe palm fruits to denature and facilitates stripping of the fruits fromthe bunch stalks. The palm fruitlets are discharged into vesselscommonly referred to as digesters, whereby a digested mash of palmfruits are produced under controlled temperature. The digested mash isthen pressed, e.g. by using a screw press for subsequent recovery ofpalm oil. The crude palm oil may be subjected to screening, e.g. toremove coarse fibers, and then to a clarification process to separateoil from water, cell debris and any remaining fibrous material.

Palm fruit mesocarp contains large amounts of oil present as oildroplets within the mesocarp cells. Generally, the oil extraction rate(OER), which is a measure of the amount of extracted oil relative to theweight of the palm fruits is within the range of 20-24%, depending e.g.on fruit quality, and is subject to seasonal variation. In general, thepalm oil milling process has been carefully optimized at each mill tominimize oil losses to the extent possible but there is still a strongincentive to improve the OER.

WO 2012/011130 discloses an enzyme composition (with exocellulolytic,pectinolytic, mammanolytic and glucanoloytic activity) used in a processfor palm oil extraction.

There remains a need for improvement of processes for wet milling orpalm oil extraction.

SUMMARY OF THE INVENTION

The invention provides a process for treating crop kernels, comprisingthe steps of: a) soaking kernels in water to produce soaked kernels; b)grinding the soaked kernels; c) and treating the soaked kernels or afraction of said corn kernels in the presence of an effective amount ofa polypeptide having GH62 arabinofuranosidase activity and/or apolypeptide having GH10 xylanase activity; wherein step c) is performedbefore, during or after step b).

In one embodiment, the process of the present invention furthercomprising fiber washing step.

In one embodiment, the process of the present invention furthercomprising starch gluten separation step and starch washing step.

In one embodiment, the invention provides a process metioned abovewherein step c) is performed during fiber washing step.

In one embodiment, the invention provides a process metioned abovewherein the soaking is performed in the presence of between 0.01-1%,preferably 0.05-0.3%, especially 0.1% SO₂ and/or NaHSO₃.

In one embodiment, the invention provides a process metioned abovewherein the crop kernels are from corn (maize), rice, barley, sorghumbean, or fruit hulls, or wheat.

In one embodiment, the invention provides a process metioned abovefurther comprising treating the soaked crop kernels or a fraction ofsaid corp kernels in the presence of one or more cellulolytic enzyme(s),preferably the one or more hydrolytic enzymes is expressed in anorganism, such as Trichoderma reesei.

In one embodiment, the invention provides a process metioned abovewherein said corp kernels or a fraction of said corp kernels is admixedwith said one or more hydrolytic enzymes, preferably the one or morehydrolytic enzymes is expressed in an organism, such as Trichodermareesei.

In one embodiment, the invention provides a process metioned abovewherein comprising treating the soaked crop kernels or a fraction ofsaid corp kernels in the presence of a polypeptide having GH30 xylanaseactivity.

In one embodiment, the invention provides a process metioned abovefurther comprising treating the soaked crop kernels or a fraction ofsaid corp kernels in the presence of an enzyme selected from the groupconsisting of a cellulolytic enzyme or a cellulase, an endoglucanase, aprotease, a cellobiohydrolase I, a cellobiohydrolase II, a GH61polypeptide, or a combination thereof.

In one embodiment, the invention provides a process metioned abovewherein the GH62 polypeptide having arabinofuranosidase activity isderived from a strain of genus Aspergillus, such as a strain ofAspergillus niger.

In one embodiment, the invention provides a process metioned abovewherein the polypeptide having GH62 arabinofuranosidase activity isselected from the group consisting of:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 1 or the maturepolypeptide of SEQ ID NO: 3;

(b) a variant of the mature polypeptide of SEQ ID NO: 1 or the maturepolypeptide of SEQ ID NO: 3 comprising a substitution, deletion, and/orinsertion at one or more (several) positions.

In one embodiment, the invention provides a process metioned abovewherein the polypeptide having GH10 xylanase activity is derived from astrain of the genus Aspergillus, such as a strain of Aspergillus niger.

In one embodiment, the invention provides a process metioned abovewherein the polypeptide having GH10 xylanase activity is selected fromthe group consisting of:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 4;

(b) a variant of the mature polypeptide of SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (several)positions.

In one embodiment, the invention provides a process metioned abovewherein the polypeptide having GH30 xylanase activity is selected fromthe group consisting of:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 6;

(b) a variant of the mature polypeptide of SEQ ID NO: 6 comprising asubstitution, deletion, and/or insertion at one or more (several)positions.

In one embodiment, the invention provides a process metioned abovewherein the polypeptide having GH30 xylanase activity is derived from astrain of the genus Bacillus, such as a strain of Bacillus subtilis.

In one embodiment, the invention provides a process metioned abovewherein the fiber washing step comprise a space configured to provide atotal retention time in the fiber washing system of at least 0.5 hour,preferably at least 2 hours, most preferably at least 4 hours.

The invention also provides a polypeptide having GH62arabinofuranosidase activity, selected from the group consisting of:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 1;

(b) a variant of the mature polypeptide of SEQ ID NO: 1 comprising asubstitution, deletion, and/or insertion at one or more (several)positions;

(c) a polypeptide encoded by a polynucleotide having at least 85%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 2 or the cDNA sequence thereof.

In one embodiment, the polypeptide having GH62 arabinofuranosidaseactivity of the invention is derived from a strain of the genusAspergillus, such as a strain of Aspergillus niger.

The invention also provides a polypeptide having GH10 xylanase activity,selected from the group consisting of:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 4;

(b) a variant of the mature polypeptide of SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (several)positions;

(c) a polypeptide encoded by a polynucleotide having at least 85%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 5 or the cDNA sequence thereof.

In one embodiment, the polypeptide having GH10 xylanase activity of theinvention is derived from a strain of the genus Aspergillus, such as astrain of Aspergillus niger.

The invention also provides a process for extraction or separation ofcrude palm oil, comprising the steps of: contacting a substratecomprising palm oil with an enzyme composition, extracting or separatingthe crude palm oil; wherein the enzyme composition comprises a GH10xylanase and a GH62 arabinofuranosidase.

In one embodiment, the GH10 xylanase of the present invention and theGH62 arabinofuranosidase of the present invention are defined in any oneof the preceding embodiments.

The invention also provides a use of a polypeptide having GH62arabinofuranosidase activity, a polypeptide having GH10 xylanaseactivity, or a polypeptide having GH30 xylanase activity, to improve thetotal starch yield and/or gluten yield from corn kernels in a process asdefined in any one of the preceding embodiments or to improve oil yieldfrom crude palm oil in a process defined in any one of the precedingembodiments, wherein, preferably the said polypeptides are defined inany one of the preceding embodiments.

The invention also provides an enzyme composition comprising orconsisting of a GH62 arabinofuranosidase, a GH10 xylanase, and/or a GH30xylanase, preferably, the GH62 arabinofuranosidase, the GH10 xylanaseand/or the GH30 xylanase is defined in any one of the precedingembodiments.

In one embodiment, the enzyme composition of the present inventionfurther comprising of one or more hydrolytic enzymes, preferably one ormore cellulolytic enzyme, preferably, the one or more cellulolyticenzymes is expressed in an organism, such as Trichoderma reesei.

Definitions

Arabinofuranosidase: The term “arabinofuranosidase” means analpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) thatcatalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,2)- and/or (1,5)-linkages, arabinoxylans, andarabinogalactans. Alpha-L-arabinofuranosidase is also known asarabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase,alpha-L-arabinofuranoside hydrolase, L-arabinosidase, oralpha-L-arabinanase. Arabinofuranosidase activity can be determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

The arabinofuranosidase of the present invention have at least at least20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 100% of thearabinofuranosidase activity of one or more of the polypeptides selectedfrom the list consisting of SEQ ID NO: 1 and SEQ ID NO: 3.

Arabinoxylan-containing material: The term “Arabinoxylan-containingmaterial” means any material containing arabinoxylan. Arabinoxylan is ahemicellulose found in both the primary and secondary cell walls ofplants, including woods and cereal grains, consisting of copolymers oftwo pentose sugars, arabinose and xylose. The arabinoxylan chaincontains a large number of 1,4-linked xylose units. Many xylose unitsare substituted with 2-, 3- or 2,3-substituted arabinose residues.

Examples of arabinoxylan-containing material are forage, roughage, seedsand grains (either whole or prepared by crushing, milling, etc from e.g.corn, oats, rye, barley, wheat), trees or hard woods (such as poplar,willow, eucalyptus, palm, maple, birch), bamboo, herbaceous and/or woodyenergy crops, agricultural food and feed crops, animal feed products,cassava peels, cocoa pods, sugar cane, sugar beet, locust bean pulp,vegetable or fruit pomaces, wood waste, bark, shavings, sawdust, woodpulp, pulping liquor, waste paper, cardboard, construction anddemolition wood waste, industrial or municipal waste water solids orsludge, manure, by-product from brewing and/or fermentation processes,wet distillers grain, dried distillers grain, spent grain, vinasse andbagasse.

Forage as defined herein also includes roughage. Forage is fresh plantmaterial such as hay and silage from forage plants, grass and otherforage plants, grass and other forage plants, seaweed, sprouted grainsand legumes, or any combination thereof. Examples of forage plants areAlfalfa (Lucerne), birdsfoot trefoil, brassica (e.g. kale, rapeseed(canola), rutabaga (swede), turnip), clover (e.g. alsike clover, redclover, subterranean clover, white clover), grass (e.g. Bermuda grass,brome, false oat grass, fescue, heath grass, meadow grasses, miscanthus,orchard grass, ryegrass, switchgrass, Timothy-grass), corn (maize),hemp, millet, barley, oats, rye, sorghum, soybeans and wheat andvegetables such as beets. Crops suitable for ensilage are the ordinarygrasses, clovers, alfalfa, vetches, oats, rye and maize. Forage furtherincludes crop residues from grain production (such as corn stover; strawfrom wheat, barley, oat, rye and other grains); residues from vegetableslike beet tops; residues from oilseed production like stems and leavesform soy beans, rapeseed and other legumes; and fractions from therefining of grains for animal or human consumption or from fuelproduction or other industries.

Roughage is generally dry plant material with high levels of fiber, suchas fiber, bran, husks from seeds and grains and crop residues (such asstover, copra, straw, chaff, sugar beet waste).

Preferred sources of arabinoxylan-containing materials are forage,roughage, seeds and grains, sugar cane, sugar beet and wood pulp.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyses the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. Xylanase activity can be determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

In one aspect, the GH10 xylanase of the present invention have at least20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 100% of the xylanaseactivity of the mature polypeptide of SEQ ID NO:4.

In one aspect, the GH30 xylanase of the present invention have at least20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 100% of the xylanaseactivity of the mature polypeptide of SEQ ID NO: 6.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teed, 1997, Trends in Biotechnology 15: 160-167; Teed et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity canbe determined according to the procedures described by Lever et al.,1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzymeactivity can be measured using insoluble substrates, including WhatmanNo 1 filter paper, microcrystalline cellulose, bacterial cellulose,algal cellulose, cotton, pretreated lignocellulose, etc. The most commontotal cellulolytic activity assay is the filter paper assay usingWhatman No 1 filter paper as the substrate. The assay was established bythe International Union of Pure and Applied Chemistry (IUPAC) (Ghose,1987, Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increasein production/release of sugars during hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in pretreated cornstover (PCS) (or other pretreated cellulosic material) for 3-7 days at asuitable temperature such as 40° C.-80° C., e.g., 40° C., 45° C., 50°C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C., and a suitablepH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,or 9.0, compared to a control hydrolysis without addition ofcellulolytic enzyme protein. Typical conditions are 1 ml reactions,washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodiumacetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugaranalysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories,Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. Cellulose is a homopolymer of anyhdrocellobioseand thus a linear beta-(1-4)-D-glucan, while hemicelluloses include avariety of compounds, such as xylans, xyloglucans, arabinoxylans, andmannans in complex branched structures with a spectrum of substituents.Although generally polymorphous, cellulose is found in plant tissueprimarily as an insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Crop kernels: The term “crop kernels” includes kernels from, e.g., corn(maize), rice, barley, sorghum bean, fruit hulls, and wheat. Cornkernels are exemplary. A variety of corn kernels are known, including,e.g., dent corn, flint corn, pod corn, striped maize, sweet corn, waxycorn and the like. In an embodiment, the corn kernel is yellow dent cornkernel. Yellow dent corn kernel has an outer covering referred to as the“Pericarp” that protects the germ in the kernels. It resists water andwater vapour and is undesirable to insects and microorganisms. The onlyarea of the kernels not covered by the “Pericarp” is the “Tip Cap”,which is the attachment point of the kernel to the cob.

Dry solids: The term “dry solids” is the total solids of a slurry inpercent on a dry weight basis.

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). Endoglucanase activity can also bedetermined using carboxymethyl cellulose (CMC) as substrate according tothe procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5,40° C.

Protease: The term “proteolytic enzyme” or “protease” means one or more(e.g., several) enzymes that break down the amide bond of a protein byhydrolysis of the peptide bonds that link amino acids together in apolypeptide chain. A protease may include, e.g., a metalloprotease, atrypsin-like serine protease, a subtilisin-like serine protease, andaspartic protease.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide, wherein the fragment has enzymeactivity. In one aspect, a fragment contains at least 85%, e.g., atleast 90% or at least 95% of the amino acid residues of the maturepolypeptide of an enzyme.

Germ: The “Germ” is the only living part of the corn kernel. It containsthe essential genetic information, enzymes, vitamins, and minerals forthe kernel to grow into a corn plant. In yellow dent corn, about 25percent of the germ is corn oil. The endosperm covered or surrounded bythe germ comprises about 82 percent of the kernel dry weight and is thesource of energy (starch) and protein for the germinating seed. Thereare two types of endosperm, soft and hard. In the hard endosperm, starchis packed tightly together. In the soft endosperm, the starch is loose.

Grind or grinding: The term “grinding” means any process that breaks thepericarp and opens the crop kernel.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Milled: The term “milled” refers to plant material which has been brokendown into smaller particles, e.g., by crushing, fractionating, grinding,pulverizing, etc.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc.

In one aspect, the mature polypeptide of SEQ ID NO: 1 consists of aminoacids 27 to 332, amino acids 1 to 26 of SEQ ID NO: 1 being a signalpeptide.

In one aspect, the mature polypeptide of SEQ ID NO: 3 consists of aminoacids 27 to 332, amino acids 1 to 26 of SEQ ID NO: 3 being a signalpeptide.

In one aspect, the mature polypeptide of SEQ ID NO: 4 consists of aminoacids 20 to 319, amino acids 1 to 19 of SEQ ID NO: 4 being a signalpeptide.

In one aspect, the mature polypeptide of SEQ ID NO: 6 consists of aminoacids 27 to 417, amino acids 1 to 26 of SEQ ID NO: 6 being a signalpeptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptide.

In one aspect, the mature polypeptide coding sequence of a GH62arabinofuranosidase is nucleotides 79 to 996 of SEQ ID NO: 2 or the cDNAsequence thereof.

In another aspect, the mature polypeptide coding sequence of a GH10xylanase is nucleotides 58 to 244,301 to 341,401 to 449,511 to 632,685to 830,884 to 972,1029 to 1052,1129 to 1217,1280 to 1345,1406 to 1492 ofSEQ ID NO: 5 or the cDNA sequence thereof.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Oligosaccharide: The term “oligosaccharide” is a compound having 2 to 10monosaccharide units.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. Version 6.1.0 was used. The optional parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSSversion of BLOSUM62) substitution matrix. The output of Needle labelled“longest identity” (obtained using the −nobrief option) is used as thepercent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. Version 6.1.0 was used. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4)substitution matrix. The output of Needle labelled “longest identity”(obtained using the nobrief option) is used as the percent identity andis calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment Total Numberof Gaps in Alignment)

Starch: The term “starch” means any material comprised of complexpolysaccharides of plants, composed of glucose units that occurs widelyin plant tissues in the form of storage granules, consisting of amyloseand amylopectin, and represented as (C6H10O5)n, where n is any number.

Steep or steeping: The term “steeping” means soaking the crop kernelwith water and optionally SO₂.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having arabinofuranosidase or xylanase activity.

Substantially pure polypeptide: The term “substantially purepolypeptide” means a preparation that contains at most 10%, at most 8%,at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%,and at most 0.5% by weight of other polypeptide material with which itis natively or recombinantly associated. Preferably, the polypeptide isat least 92% pure, e.g., at least 94% pure, at least 95% pure, at least96% pure, at least 97% pure, at least 98% pure, at least 99%, at least99.5% pure, and 100% pure by weight of the total polypeptide materialpresent in the preparation. The polypeptides of the present inventionare preferably in a substantially pure form. This can be accomplished,for example, by preparing the polypeptide by well-known recombinantmethods or by classical purification methods.

Variant: The term “variant” means a polypeptide having xylanase orarabinofuranosidase activity comprising an alteration, i.e., asubstitution, insertion, and/or deletion of one or more (several) aminoacid residues at one or more (several) positions. A substitution means areplacement of an amino acid occupying a position with a different aminoacid; a deletion means removal of an amino acid occupying a position;and an insertion means adding 1-3 amino acids adjacent to an amino acidoccupying a position.

Wet milling benefit: The term “wet milling benefit” means one or more ofimproved starch yield and/or purity, improved gluten quality and/oryield, improved fiber, gluten, or steep water filtration, dewatering andevaporation, easier germ separation and/or better post-saccharificationfiltration, and process energy savings thereof.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrmann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. A common total xylanolytic activity assay is based onproduction of reducing sugars from polymeric 4-O-methyl glucuronoxylanas described in Bailey et al., 1992, Interlaboratory testing of methodsfor assay of xylanase activity, Journal of Biotechnology 23(3): 257-270.Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan assubstrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μmole of azurineproduced per minute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan assubstrate in 200 mM sodium phosphate pH 6.

Xylan degrading activity can be determined by measuring the increase inhydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo.,USA) by xylan-degrading enzyme(s) under the following typicalconditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg ofxylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50° C.,24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH)assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

Crude oil: The term “crude oil” (also called a non-degummed oil) refersto a pressed or extracted oil or a mixture thereof. In the presentcontext, it is to be understood that the oil is palm oil, in particularun-refined palm oil. In particular, the term “crude oil” refers to theeffluent from the screw press of a palm oil mill; i.e. to the mixture ofoil and water pressed out of the palm fruit mash, before it has beensubject to clarification and separation of oil from water. The crudepalm oil is also designated CPO. Crude palm oil comprises water.

Digestion: The term “digestion” refers to a process where the substratecomprising palm oil is kept at a temperature in the range of 65-85° C.for disintegrating the substrate and releasing palm oil from themesocarp. The digestion can be carried out in a digestion tank and/or aprecooker tank equipped with baffles. During the digestion, thesubstrate comprising palm oil e.g. the palm fruitlets are disintegratedand oil released from the mesocarp. According to the invention thesubstrate comprising palm oil can be contacted with the enzymecomposition before or during the digestion.

Oil extraction rate (OER): “Oil extraction rate (OER)” may be defined asby Chang et al., oil palm Industry economic journal, volume 3, 2003[9].Chang et al. defines the Oil extract rate as ratio of oil recovered andFresh fruit branch (FFB) times 100, the mathematical formula is:OER=(weight of oil recovered/weight of FFB processed)×100

Palm oil mill effluent (POME): Palm oil mill effluent (POME) is thewaste water discharged e.g. from the sterilization process, crude oilclarification process.

Palm press liquid: The term “palm press liquid” refers to the liquiddischarged from the pressing of the substrate comprising palm oil. Palmpress liquid is not a crude palm oil and water has not been separatedfrom the palm press liquid.

Nomenclature

For purposes of the present invention, the nomenclature [Y/F] means thatthe amino acid at this position may be a tyrosine (Try, Y) or aphenylalanine (Phe, F). Likewise the nomenclature [V/G/A/I] means thatthe amino acid at this position may be a valine (Val, V), glycine (Gly,G), alanine (Ala, A) or isoleucine (Ile, I), and so forth for othercombinations as described herein. Unless otherwise limited further, theamino acid X is defined such that it may be any of the 20 natural aminoacids.

DETAILED DESCRIPTION OF THE INVENTION

Milling Process

It is an object of the invention to provide improved processes oftreating crop kernels to provide starch of high quality.

In one embodiment, the enzyme compositions useful in the processes ofthe invention provide benefits including, improving starch yield and/orpurity, improving gluten quality and/or yield, improving fiber, gluten,or steep water filtration, dewatering and evaporation, easier germseparation and/or better post-saccharification filtration, and processenergy savings thereof.

Moreover, the present inventors have surprisingly found that the enzymesuseful according to the invention provide reduction in fiber mass andlower protein content of the fiber due to better separation of bothstarch and protein fractions from the fiber fraction. Separating starchand gluten from fiber is valuable to the industry because fiber is theleast valuable product of the wet milling process, and higher puritystarch and protein is desirable.

Surprisingly, the present inventors have discovered that replacing someof the protease activity in an enzyme composition can provide animprovement over an otherwise similar composition containingpredominantly protease activity alone. This can provide a benefit to theindustry, e.g., on the basis of cost and ease of use.

The kernels are milled in order to open up the structure and to allowfurther processing and to separate the kernels into the four mainconstituents: starch, germ, fiber and protein.

In one embodiment, a wet milling process is used. Wet milling gives avery good separation of germ and meal (starch granules and protein) andis often applied at locations where there is a parallel production ofsyrups.

The inventors of the present invention have surprisingly found that thequality of the starch final product may be improved by treating cropkernels in the processes as described herein.

The processes of the invention result in comparison to traditionalprocesses in a higher starch quality, in that the final starch productis more pure and/or a higher yield is obtained and/or less process timeis used. Another advantage may be that the amount of chemicals, such asSO2 and NaHSO3, which need to be used, may be reduced or even fullyremoved.

Wet Milling

Starch is formed within plant cells as tiny granules insoluble in water.When put in cold water, the starch granules may absorb a small amount ofthe liquid and swell. At temperatures up to about 50° C. to 75° C. theswelling may be reversible. However, with higher temperatures anirreversible swelling called “gelatinization” begins. Granular starch tobe processed according to the present invention may be a crudestarch-containing material comprising (e.g., milled) whole grainsincluding non-starch fractions such as germ residues and fibers. The rawmaterial, such as whole grains, may be reduced in particle size, e.g.,by wet milling, in order to open up the structure and allowing forfurther processing. Wet milling gives a good separation of germ and meal(starch granules and protein) and is often applied at locations wherethe starch hydrolyzate is used in the production of, e.g., syrups.

In an embodiment, the particle size is reduced to between 0.05-3.0 mm,preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%,more preferably at least 70%, even more preferably at least 90% of thestarch-containing material fits through a sieve with a 0.05-3.0 mmscreen, preferably 0.1-0.5 mm screen.

More particularly, degradation of the kernels of corn and other cropkernels into starch suitable for conversion of starch into mono- andoligo-saccharides, ethanol, sweeteners, etc. consists essentially offour steps:

1. Steeping and germ separation,

2. Fiber washing and drying,

3. Starch gluten separation, and

4. Starch washing.

1. Steeping and Germ Separation

Corn kernels are softened by soaking in water for between about 30minutes to about 48 hours, preferably 30 minutes to about 15 hours, suchas about 1 hour to about 6 hours at a temperature of about 50° C., suchas between about 45° C. to 60° C. During steeping, the kernels absorbwater, increasing their moisture levels from 15 percent to 45 percentand more than doubling in size. The optional addition of e.g. 0.1percent sulfur dioxide (SO2) and/or NaHSO3 to the water preventsexcessive bacteria growth in the warm environment. As the corn swellsand softens, the mild acidity of the steepwater begins to loosen thegluten bonds within the corn and release the starch. After the cornkernels are steeped they are cracked open to release the germ. The germcontains the valuable corn oil. The germ is separated from the heavierdensity mixture of starch, hulls and fiber essentially by “floating” thegerm segment free of the other substances under closely controlledconditions. This method serves to eliminate any adverse effect of tracesof corn oil in later processing steps.

In an embodiment of the invention the kernels are soaked in water for2-10 hours, preferably about 3-5 hours at a temperature in the rangebetween 40 and 60° C., preferably around 50° C.

In one embodiment, 0.01-1%, preferably 0.05-0.3%, especially 0.1% SO₂and/or NaHSO₃ may be added during soaking.

2. Fiber Washing and Drying

To get maximum starch recovery, while keeping any fiber in the finalproduct to an absolute minimum, it is necessary to wash the free starchfrom the fiber during processing. The fiber is collected, slurried andscreened to reclaim any residual starch or protein.

3. Starch Gluten Separation

The starch-gluten suspension from the fiber-washing step, called millstarch, is separated into starch and gluten. Gluten has a low densitycompared to starch. By passing mill starch through a centrifuge, thegluten is readily spun out.

4. Starch Washing

The starch slurry from the starch separation step contains someinsoluble protein and much of solubles. They have to be removed before atop quality starch (high purity starch) can be made. The starch, withjust one or two percent protein remaining, is diluted, washed 8 to 14times, re-diluted and washed again in hydroclones to remove the lasttrace of protein and produce high quality starch, typically more than99.5% pure.

Products

Wet milling can be used to produce, without limitation, corn steepliquor, corn gluten feed, germ, corn oil, corn gluten meal, corn starch,modified corn starch, syrups such as corn syrup, and corn ethanol.

Palm Oil Extraction

The present invention also provides a process for enzyme assistedextraction of crude palm oil from a substrate comprising palm oil. Thesubstrate comprising palm oil can be selected from the group consistingof palm fruitlets, pressed palm fruit liquid, mashed or partly mashedpalm fruitlets. The inventors have found that by using a GH10 xylanaseon the substrate comprising palm oil, the oil extraction rate (OER) canbe increased.

The invention concerns a process for extraction or separation of crudepalm oil (CPO), comprising the steps of:

i) contacting a substrate comprising palm oil with an enzymecomposition,

ii) extracting or separating the crude palm oil (CPO)

wherein the enzyme composition comprises a GH10 xylanase and a GH62arabinofuranosidase.

In one embodiment of the invention, the substrate comprising palm oil ispalm fruitlets, which comprise oil in the mesocarp of the fruit. Thepalm fruitlets are contacted with the enzyme composition. In oneembodiment, the substrate is palm fruitlets, which are mashed or partlymashed and contacted with the enzyme composition. This increasesavailability of mesocarp cells and thereby enhances enzyme activity onthe mesocarp cells. In one embodiment, the substrate comprising palm oilis crude palm oil which is contacted with the enzyme composition. In thevarious aspects and embodiments of the invention the substrate, whichcomprises palm oil may be a substrate which also comprises fiber, inparticular fiber from the mescocarp of palm fruitlets.

In one embodiment of the invention the substrate comprising palm oil issterilized before being contacted with the enzyme composition. Palmfruits grow in large bunches and needs to be stripped from the bunchstalks before being contacted with the enzyme composition. Steamsterilization of the fresh fruit bunches facilitates fruits beingstripped from bunches to give the palm fruitlet. The sterilization stephas several advantages one being that it softens the fruit mesocarp forsubsequent digestion. A further advantage is that the quality of thefinal palm oil product is better if the palm fruits are stripped fromthe bunch stalks.

The sterilization can be a batch sterilization or a continuoussterilization. The sterilization process can be carried out at atemperature of 100° C.-150° C. In one embodiment of the invention, thepressure is reduced during the sterilization procedure.

After the sterilization, the palm fruitlets are stripped from the bunchstalks. Stripping or threshing can be carried out in a mechanized systemhaving a rotating drum or fixed drum equipped with rotary beater barswhich detach the fruit from the bunch and leaves the spikelets on thestem. The stripped palm fruitlets can be contacted with the enzymecomposition according to the invention.

In one embodiment of the invention, the substrate comprising palm oil issubjected to digestion before extracting the crude palm oil. Thestripped palm fruitlets can be transported into a digester by one ormore transportation means, e.g. a conveyor belt. In the digester, thefruitlets are further heated in order to loosen the pericarp. Thedigester is typically a steam heated vessel, which has rotating shaftsto which stirring arms are attached or is equipped with baffles. Thefruitlets are rotated, causing the loosening of the pericarps from thenuts and degradation of the mesocarp. The digester is a continuousprocess where the digester is kept full and as the digested fruit isdrawn out, freshly stripped fruits are brought in.

In one embodiment of the invention, the first part of the digestion iscarried out in a precooker. The substrate may be held at a temperaturewithin the range of 65-85° C. for some time and then transferred to thedigester tank.

Polypeptides Having GH62 Arabinofuranosidase Activity

Preferred embodiments of the aspect of the invention relating to theGH62 polypeptide having arabinofuranosidase activity are disclosedherein below.

In an embodiment, the polypeptide having GH62 arabinofuranosidaseactivity of the present invention, is selected from the group consistingof:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 1;

(b) a variant of the mature polypeptide of SEQ ID NO: 1 comprising asubstitution, deletion, and/or insertion at one or more (several)positions;

(c) a polypeptide encoded by a polynucleotide having at least 85%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 2 or the cDNA sequence thereof.

In an embodiment, the polypeptide having GH62 arabinofuranosidaseactivity of the present invention, is selected from the group consistingof:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 3;

(b) a variant of the mature polypeptide of SEQ ID NO: 3 comprising asubstitution, deletion, and/or insertion at one or more (several)positions.

In one aspect, the polypeptide differs by up to 10 amino acids, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide. In anotherembodiment, the present invention relates to variants of the maturepolypeptide comprising a substitution, deletion, and/or insertion at oneor more (e.g., several) positions. In an embodiment, the number of aminoacid substitutions, deletions and/or insertions introduced into themature polypeptide is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function.

A polypeptide having arabinofuranosidase activity may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a polynucleotide is producedby the source or by a strain in which the polynucleotide from the sourcehas been inserted. In one aspect, the polypeptide obtained from a givensource is secreted extracellularly.

The polypeptide may be a fungal polypeptide. In one embodiment, thepolypeptide is from a fungus of the order Eurotiales, or from the familyAspergillaceae, or from the genus Aspergillus or from the speciesAspergillus clavatus or Aspergillus wentii or Aspergillus niger.

In one embodiment, the GH62 arabinofuranosidase is derived from a strainof the genus Aspergillus, such as a strain of Aspergillus niger.

In one embodiment, the polypeptide is from a fungus of the orderEurotiales, or from the family Aspergillaceae, or from the genusNeosartorya or from the species Neosartorya fischeri.

In one embodiment, the polypeptide is from a fungus of the orderEurotiales, or from the family Trichocomaceae, or from the genusTalaromyces or from the species Talaromyces pinophilus.

The polypeptide may be a bacterial polypeptide. In one embodiment, thepolypeptide is from a bacterium of the order Actinomycetales, or fromthe family Streptomycetaceae, or from the genus Streptomyces or from thespecies Streptomyces nitrosporeus or Streptomyces beijiangensis.

In one embodiment, the polypeptide is from a bacterium of the orderActinomycetales, or from the family Streptosporangiaceae, or from thegenus Streptosporangium or from the species Streptosporangium sp-60756.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polypeptides Having GH10 Xylanase Activity

Exemplary embodiments relating to the GH10 polypeptide having xylanaseactivity are disclosed herein below.

In an embodiment, the polypeptide having GH10 xylanase activity,selected from the group consisting of:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 4;

(b) a variant of the mature polypeptide of SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (several)positions;

(c) a polypeptide encoded by a polynucleotide having at least 85%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 5 or the cDNA sequence thereof.

In one aspect, the polypeptide differs by up to 10 amino acids, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide. In anotherembodiment, the present invention relates to variants of the maturepolypeptide comprising a substitution, deletion, and/or insertion at oneor more (e.g., several) positions. In an embodiment, the number of aminoacid substitutions, deletions and/or insertions introduced into themature polypeptide is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function.

A polypeptide having xylanase activity of the present invention (GH10xylanase) may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a polynucleotide is produced by the source or by a strain in whichthe polynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a Talaromyces polypeptide.

In another embodiment, the polypeptide is a Talaromyces leycettanuspolypeptide, e.g., a polypeptide obtained from Talaromyces leycettanusStrain CBS398.68.

The polypeptide may be an Aspergillus polypeptide. In anotherembodiment, the polypeptide is an Aspergillus niger polypeptide,

In one embodiment, the GH10 xylanase is derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus niger.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polypeptides Having GH30 Xylanase Activity

GH30 polypeptide refers to a polypeptide with enzyme activity, thepolypeptide being classified as member of the Glycoside hydrolase family30 in the database of Carbohydrate-Active enZYmes (CAZymes)(http://www.cazy.org/).

In one embodiment, the polypeptide having GH30 xylanase activity isselected from the group wherein the polypeptide having GH30 xylanaseactivity is selected from the group consisting of:

(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 6;

(b) a variant of the mature polypeptide of SEQ ID NO: 6 comprising asubstitution, deletion, and/or insertion at one or more (several)positions.

In one aspect, the polypeptide differs by up to 10 amino acids, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide. In anotherembodiment, the present invention relates to variants of the maturepolypeptide comprising a substitution, deletion, and/or insertion at oneor more (e.g., several) positions. In an embodiment, the number of aminoacid substitutions, deletions and/or insertions introduced into themature polypeptide is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function.

A polypeptide having xylanase activity of the present invention (GH30xylanase) may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a polynucleotide is produced by the source or by a strain in whichthe polynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

In one embodiment, the polypeptide having GH30 xylanase activity isderived from a strain of the genus Bacillus, such as a strain ofBacillus subtilis.

The polypeptide may be a bacterial polypeptide. In one embodiment, thepolypeptide may be a Bacillus polypeptide. In another embodiment, thepolypeptide is a Bacillus subtilis polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Cellulolytic Composition

Exemplary cellulolytic compositions are as described in e.g., WO2015/081139 and PCT/US2015/034179.

In an embodiment, the cellulolytic composition is derived from a strainof Trichoderma, such as a strain of Trichoderma reesei; a strain ofHumicola, such as a strain of Humicola insolens, and/or a strain ofChrysosporium, such as a strain of Chrysosporium lucknowense.

In a preferred embodiment, the cellulolytic composition is derived froma strain of Trichoderma reesei.

In a preferred embodiment, the cellulolytic composition is a Trichodermareesei cellulase preparation.

In an embodiment, the cellulolytic composition comprises a Trichodermareesei cellulase preparation containing Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637) and Thermoascusaurantiacus GH61A polypeptide (WO 2005/074656).

In an embodiment, the cellulolytic composition comprises a Trichodermareesei cellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity (WO2005/074656) and Aspergillus oryzae beta-glucosidase fusion protein (WO2008/057637).

In another embodiment, the cellulolytic composition comprises aTrichoderma reesei cellulolytic enzyme composition, further comprisingThermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity (Sequence Number 2 in WO 2005/074656) and Aspergillus fumigatusbeta-glucosidase (Sequence Number 2 of WO 2005/047499).

In another embodiment, the cellulolytic composition comprises aTrichoderma reesei cellulolytic enzyme composition, further comprisingPenicillium emersonii GH61A polypeptide having cellulolytic enhancingactivity disclosed in WO 2011/041397, Aspergillus fumigatusbeta-glucosidase (Sequence Number 2 of WO 2005/047499) or a variantthereof with the following substitutions: F100D, S283G, N456E, F512Y.

In an embodiment, the cellulolytic composition is derived fromTrichoderma reesei RutC30.

In an embodiment, the cellulolytic composition comprises a Trichodermareesei cellulase preparation containing Trichophaea saccata GH10xylanase (WO 2011/057083) and Talaromyces emersonii beta-xylosidase.

Enzyme Composition

The present invention also provides an enzyme composition comprising ofa GH62 arabinofuranosidase, a GH10 xylanase, and/or a GH30 xylanase.

In an embodiment, the enzyme composition of the present inventionfurther comprises one or more hydrolytic enzymes, preferably one or morecellulolytic enzyme, preferably, the one or more cellulolytic enzymes isexpressed in an organism, such as Trichoderma reesei.

In an embodiment, the enzyme composition of the present inventionfurther comprises a cellulolytic composition.

Preferably, the compositions are enriched in the polypeptides usefulaccording to the invention. The term “enriched” indicates that theenzymatic activity of the composition has been increased, e.g., with anenrichment factor of at least 1.1, such as at least 1.2, at least 1.3,at least 1.4, at least 1.5, at least 2.0, at least 3.0, at least 4.0, atleast 5.0, at least 10. In an embodiment, the composition comprises thepolypeptides of the first aspect of the invention and one or moreformulating agents, as described in the ‘formulating agent’ sectionbelow.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition. Sucha composition may further comprise a formulating agent, as described inthe ‘formulating agent’ section below. Alternatively, the compositionsmay comprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of phytase,xylanase, galactanase, alpha-galactosidase, protease, phospholipase,glucoronidase, lysophospholipase, amylase, beta-glucanase,arabinofuranosidase, beta-xylosidase, endo-1,4-beta-xylanase acetylxylan esterase, feruloyl esterase, cellulase, cellobiohydrolase,beta-glycosidase, pullulanase, or any mixture thereof. Additionalcellulolytic activities are particularly contemplated, as furtheroutlined below.

Where arabinofuranosidase and xylanase activity are contemplated, it isat present contemplated that the xylanase is used in one or more of thefollowing amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20;0.1-1; 0.2-2; 0.5-5; or 1-10 wherein all these ranges are mg xylanaseprotein per kg substrate (ppm). It is at present contemplated that thearabinofuranosidase is administered in one or more of the followingamounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1;0.2-2; 0.5-5; or 1-10 wherein all these ranges are mgarabinofuranosidase protein per kg substrate (ppm). It is furthercontemplated that the ratio of the GH10 xylanase to GH62arabinofuranosidase is in the range of 100:1 to 1:100xylanase:arabinofuranosidase such as the ranges 50:1 to 1:50, 50:1 to1:10, 25:1 to 1:5, 10:1 to 1:2 or such as 10:1 to 1:50, 5:1 to 1:25, 2:1to 1:10 xylanase:arabinofuranosidase.

Formulating Agent

The enzyme of the invention may be formulated as a liquid or a solid.For a liquid formulation, the formulating agent may comprise a polyol(such as e.g. glycerol, ethylene glycol or propylene glycol), a salt(such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or asugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, andsorbitol). Thus in one embodiment, the composition is a liquidcomposition comprising the polypeptide of the invention and one or moreformulating agents selected from the list consisting of glycerol,ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, sodiumchloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose,and sorbitol.

For a solid formulation, the formulation may be for example as agranule, spray dried powder or agglomerate. The formulating agent maycomprise a salt (organic or inorganic zinc, sodium, potassium or calciumsalts such as e.g. such as calcium acetate, calcium benzoate, calciumcarbonate, calcium chloride, calcium citrate, calcium sorbate, calciumsulfate, potassium acetate, potassium benzoate, potassium carbonate,potassium chloride, potassium citrate, potassium sorbate, potassiumsulfate, sodium acetate, sodium benzoate, sodium carbonate, sodiumchloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate,zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zincsulfate), starch or a sugar or sugar derivative (such as e.g. sucrose,dextrin, glucose, lactose, sorbitol).

In an embodiment, the solid composition is in granulated form. Thegranule may have a matrix structure where the components are mixedhomogeneously. However, the granule typically comprises a core particleand one or more coatings, which typically are salt and/or wax coatings.The core particle can either be a homogeneous blend of xylanase of theinvention optionally combined with one or more additional enzymes andoptionally together with one or more salts or an inert particle with thexylanase of the invention optionally combined with one or moreadditional enzymes applied onto it.

In an embodiment, the material of the core particles are selected fromthe group consisting of inorganic salts (such as calcium acetate,calcium benzoate, calcium carbonate, calcium chloride, calcium citrate,calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate,potassium carbonate, potassium chloride, potassium citrate, potassiumsorbate, potassium sulfate, sodium acetate, sodium benzoate, sodiumcarbonate, sodium chloride, sodium citrate, sodium sulfate, zincacetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate,zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (suchas e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugarderivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol),small organic molecules, starch, flour, cellulose and minerals.

The salt coating is typically at least 1 μm thick and can either be oneparticular salt or a mixture of salts, such as Na₂SO₄, K₂SO₄, MgSO₄and/or sodium citrate. Other examples are those described in e.g. WO2008/017659, WO 2006/034710, WO 1997/05245, WO 1998/54980, WO1998/55599, WO 2000/70034 or polymer coating such as described in WO2001/00042.

Enzymatic Amount

Enzymes may be added in an effective amount during wet milling process,which can be adjusted according to the practitioner and particularprocess needs. In general, enzyme may be present in an amount of0.0001-1 mg enzyme protein per g dry solids (DS) kernels, such as0.001-0.1 mg enzyme protein per g DS kernels. In particular embodiments,the enzyme may be present in an amount of, e.g., 1 μg, 2.5 μg, 5 μg, 10μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 75 μg, 100 μg, 125μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350μg, 375 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg enzyme protein per g DSkernels.

In some embodiments of palm oil extraction, the enzyme(s) are dosed atamounts corresponding to 10-1000 ppm, such as 20-1000 ppm, 30-1000 ppm,40-1000 ppm, 50-1000 ppm, 100-1000 ppm, 200-1000 ppm, 100-500 ppm, suchas 200-500 ppm, 250-400 ppm or 350-1000 ppm relative to the amount ofsubstrate comprising palm oil.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Materials and Methods

Enzymes

GH62 Arabinofuranosidase A: GH62 arabinofuranosidase derived fromAspergillus niger (SEQ ID NO: 1).

GH62 Arabinofuranosidase B: GH62 arabinofuranosidase derived fromAspergillus niger (SEQ ID NO: 3).

GH10 Xylanase A: GH10 xylanase derived from Aspergillus niger (SEQ IDNO: 4).

GH30 Xylanase A: GH30 xylanase derived from Bacillus subtilis (SEQ IDNO: 6)

Celluclast 1.5 L: a commercial product comprising cellulase andavailable at Novozymes A/S.

Example 1: Cloning and Recombinant Expression of a GH62Arabinofuranosidase from Aspergillus niger

The arabinofuranosidase encoding gene with SEQ ID NO: 2 was PCRamplified from genomic DNA isolated from an Aspergillus niger strain,which was isolated from Ireland, using gene-specific primers that alsoincluded a Kozak translation initiation sequence, “CACC”, immediately 5′of the start codon. The PCR amplified product was cloned into theAspergillus expression vector pMStr57 (WO 04/032648) that had beendigested with the restriction enzymes BamHI and XhoI.

The sequence of the GH62 arabinofuranosidase encoding gene cloned in theexpression vector was confirmed, and the expression construct wastransformed into the Aspergillus oryzae strain MT3568 by the methodsdescribed in Christensen et al., 1988, Biotechnology 6, 1419-1422 and WO04/032648. Transformants were selected during regeneration fromprotoplasts based on the ability to utilize acetamide as a nitrogensource conferred by a selectable marker in the expression vector.Production of the recombinant arabinofuranosidase was evaluated byculturing the transformants in 96-well deep-well microtiter plates for 4days at 30° C. in YPG medium (WO 05/066338) and monitoringarabinofuranosidase expression by SDS-PAGE. The transformant showing thehighest level of expression in microtiter plate culture was selected andre-isolated twice under selection.

For larger-scale production of the recombinant arabinofuranosidase, theselected transformant was cultured in 500 ml baffled flasks containing150 ml of DAP-4C-1 medium (WO 12/103350). The cultures were shaken on arotary table at 150 RPM at for 4 days. The culture broth wassubsequently separated from cellular material by passage through a 0.22μm filtration unit.

Example 2: Chromatographic Purification of the RecombinantArabinofuranosidase from Aspergillus niger

pH of the filtered sample was adjusted to around pH 7.5 and 1.8Mammonium sulfate was added. The sample was applied to a 5 ml HiTrap™Phenyl (HS) column on an Akta Explorer. Prior to loading, the column hadbeen equilibrated in 5 column volumes (CV) of 50 mM HEPES+1.8M AMS(ammonium sulfate) pH 7. In order to remove unbound material, the columnwas washed with 5 CV of 50 mM HEPES+1.8M AMS pH 7. The target proteinwas eluted from the column into a 10 ml loop using 50 mM HEPES+20%isopropanol pH 7. From the loop, the sample was loaded onto a desaltingcolumn (HiPrep™ 26/10 Desalting), which had been equilibrated with 3CVof 50 mM HEPES+100 mM NaCl pH 7.0. The target protein was eluted with 50mM HEPES+100 mM NaCl pH 7.0 and relevant fractions were selected andpooled based on the chromatogram. The flow rate was 5 ml/min.

The GH62 arabinofuranosidase coding sequence and the full-length aminoacid sequence of the GH62 arabinofuranosidase are shown in SEQ ID NO: 1and SEQ ID NO: 2, respectively. Determination of the N-terminal sequencewas: KCSLPSS, which was determined by N-terminal Edman degradationSequencing.

Example 3: Genomic DNA Extraction from Aspergillus niger NN053297

The Aspergillus niger strain NN053297 was isolated from hot spring soilsamples collected from Yunnan province in 2010.

Aspergillus niger strain NN053297 was inoculated on PDA plate andincubated for 37 C for 4 days. Mycelia were collected and frozen inliquid nitrogen in a sterilized mortar and grounded with pestle to finepowders. Then the genomic DNA was extracted with Biospin Fungus GenomicDNA Extraction Kit (Bioer Technology Co. Ltd., Hangzhou, China)following the manufacturer's instruction.

Example 4: Cloning of Aspergillus niger GH10 Xylanase Gene into anAspergillus oryzae Expression Vector

Based on the DNA information from public database, oligonucleotideprimers, shown below, were designed to amplify the coding sequence ofthe Aspergillus niger GH10 xylanase. The GH10 xylanase coding sequenceand the full-length amino acid sequence are shown as SEQ ID NO: 5 & SEQID NO: 4. The primers were synthesized by Invitrogen, Beijing, China.

primer1 ACACAACTGGGGATCCACCatggttcagatcaaggtagct gcac primer2CCCTCTAGATCTCGAGctagagagcatttgcgatagcagt gta

Lowercase characters of primer1 and primer2 represent the coding regionthe gene. While bold characters represent a region homologous toinsertion sites of Aspergillus oryzae expression vector pCaHj505 asdescribed in WO2013029496. The 4 underlined letters in primer1 representthe Kozark sequence as the initiation of translation process.

The genomic DNA was prepared in Example 1. A Phusion™ High-Fidelity DNAPolymerase (Finnzymes Oy, Espoo, Finland) was used for the PCRamplification. An In-fusion CF Dry-down PCR Cloning Kit (BD Biosciences,Palo Alto, Calif., USA) was used to clone the fragment into theexpression vector pCaHj505. The expression vector pCaHj505 contained theTAKA-amylase promoter derived from Aspergillus oryzae and theAspergillus niger glucoamylase terminator elements. Furthermore pCaHj505had pUC19 derived sequences for selection and propagation in E. coli,and an amdS gene, which encoded an acetoamidase gene derived fromAspergillus nidulans for selection of an amds+Aspergillus transformant.Plasmid pCaHj505 was linearized by digestion with Bam I and Xho I,isolated by 1.0% agarose gel electrophoresis using TBE buffer, andpurified using an illustra GFX PCR DNA and Gel Band Purification Kit (GEHealthcare, Buckinghamshire, UK) following the manufacturer'sinstructions.

For the gene amplification, the PCR reaction was performed whichcontained the primer pair, primer 1 & 2, and the genomic DNA ofAspergillu niger NN053297 as the template. In brief, 20 picomoles ofeach of the primer pair were used in a PCR reaction composed of 2 μl ofgenomic DNA, 10 μl of 5× Phusion® GC Buffer (Finnzymes Oy, Espoo,Finland), 1 μl of 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6unit of PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo,Finland) in a final volume of 50 μl with deionized water. Theamplification was performed using a Peltier Thermal Cycler (MJ ResearchInc., South San Francisco, Calif., USA) programmed for denaturing at 98°C. for 1 minute; 10 cycles each of denaturing at 98° C. for 15 seconds,annealing at 68° C. for 30 seconds, with a 1° C. decrease per cycle andelongation at 72° C. for 3 minutes; 25 cycles each at 98° C. for 15seconds, 58° C. for 30 seconds, and 72° C. for 3 minutes; and a finalextension at 72° C. for 7 minutes. The heat block then went to a 4° C.soak cycle.

The PCR product, ˜1.5 kb, was purified from solution by using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit.

The purified PCR product was then ligated to the linearized vectorpCaHj505 by using In-Fusion™ Dry-down PCR Cloning Kit, resulting inp505-GH10_AnNz, in which the transcription of the of Aspergillus nigerGH10 xylanase gene was under the control of a promoter from the gene forAspergillus oryzae alpha-amylase. Briefly, for the ligation reaction,the pellet of In-Fusion Dry Down mix was suspended in 2 μl of doubledistilled H2O and 1 μl was taken to add to 0.3 μl of the linearizedvector pCaHj505 and 3.7 μl PCR product in the tube. The ligationreaction was incubated at 50° C. for 15 min.

All 5 μl of the ligation solution were used for transformation of E.coli TOP10 competent cell (TIANGEN Biotech (Beijing) Co. Ltd., Beijing,China). The ligation solution was added to 50 μl of frozen-thawcompetent cells and kept on ice for 30 minutes. Then the cells wereheat-shocked at 42° C. for 1 min, and placed on ice for 2 min. Next, 200μl of LB medium were added to the cells and incubated at 37° C. for 60min shaking at 350 rpm in a thermomixer. Finally, all the cells werespread on LB plate containing 100 ug/ml of ampicillin and incubated at37° C. overnight.

2 colonies were picked up for sequencing using 3730XL DNA Analyzers(Applied Biosystems Inc, Foster City, Calif., USA). After the sequenceswere confirmed, the colony with correct insertion was inoculated forplasmid DNA extraction with a QIAPREP® Spin Miniprep Kit (QIAGEN GmbH,Hilden, Germany) by following the manufacturer's instruction. Thus theplasmid DNA of p505-GH10_AnNz was prepared for A. oryzae transformationsin Example 5.

Example 5: Expression of the Aspergillus niger GH10 Xylanase Gene inAspergillus oryzae MT3568

Aspergillus oryzae MT3568 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. For thetransformations, 3 μg of plasmid DNA of p505-GH10_AnNz were used totransform Aspergillus oryzae MT3568. The transformation yielded severaltransformants. Four transformants were isolated and inoculated into 3 mlof Dap4C medium in 24-well plate and incubated at 30° C., 150 rpm. After3 days incubation, 20 μl of supernatant from each culture were analyzedon NuPAGE Novex 4-12% Bis-Tris Gel w/MES (Invitrogen Corporation,Carlsbad, Calif., USA) according to the manufacturer's instructions. Theresulting gel was stained with Instant Blue (Expedeon Ltd., BabrahamCambridge, UK). SDS-PAGE profiles of the cultures showed that all 4transformants had a protein band at 35 kDa. The transformant with thehighest expression level of each gene was designated as O34EQ8.

Example 6: Preparation of an Aspergillus niger GH10 Xylanase

2 slants of the expression strain 034EQ8 used for inoculation of 12flasks of 2-liter each containing 400 ml of Dap4C medium. In detail,each slant was washed with 10 ml of Dap4C medium and inoculated into 6flasks, shaking at 30 C, 80 rpm. The culture was harvested on day 4 andfiltered using a 0.22 μm DURAPORE Membrane (Millipore, Bedford, Mass.,USA). After purification the Aspergillus niger GH10 xylanase wasobtained.

Example 7: Use of Enzyme in Wet Milling Process

The amount of starch and gluten separated from fiber, after incubationwith and without enzyme, is measured by 10-g fiber assay. The 10-g fiberassay is generally described that it incubates wet fiber samplesobtained from wet-milling plant after fiber pressing and re-suspended inlactic acid buffer to 200-g slurry containing 5% dry solids of fiber,which equals to the fiber content from 100 gram dry substance of corn,in the presence of enzymes, at conditions relevant to the process (pH3.5 to 4, temperature around 52° C.) and over a time period of between 1to 4 hr. After incubation the slurry is transferred and pressed over asieve (typically 100 micron or smaller), while collecting the filtratepassing through. The fiber that retained over the sieve is pressed usinga spatula to recover as much filtrate as possible. The pressed fiber isthen transferred to a beaker containing 200-ml of water and stirred. Theslurry is passed through the 75-micron sieve and the collected filtrateis combined with the first. The pressing, washing and filtering stepsabove is repeated once more, so that a final filtrate is recovered andcombined with the first two. The combined filtrate is then vacuumfiltered, this time through a glass micro filter paper (Whatman) whichretains the insoluble solids that are released from the fiber and passedthrough the 75-micron screen. After passing 200 ml water over the filterpaper to remove any trace of solubles, the total insoluble solidsretained on the filter paper is dried and weighed. The dry weight isreported as Starch+Gluten released as percentage (w/w) of fiber drymatter of starting substrate.

Example 8: Use of GH10 Xylanase and/or GH62 Arabinofuranosidase

10-g fiber assay is performed at pH 3.8, incubating at 52° C. for 1 hourat dose of 35 ug enzyme protein per gram corn, using a blend includingCelluclast and GH10 Xylanase A, in combination with either GH62Arabinofuranosidase A or GH62 Arabinofuranosidase B. Blend consists of5% GH62 Arabinofuranosidase A or GH62 Arabinofuranosidase B, 15% of GH10Xylanase A, and the remaining 80% from Celluclast. For comparison, blendcontaining Celluclast and GH10 Xylanase A only (no GH62Arabinofuranosidase) was included. The corn fiber with 13.63% residualstarch and 10.44% residual protein was used as substrate in the fiberassay. Release of starch+gluten (dry substance) from corn fiber at thespecified doses below was measured.

TABLE 1 Dose (ug enzyme Starch + Gluten Treatments protein/g corn)Recovered No Enzyme 0  6.55% Celluclast + GH10 Xylanase A 35  8.90%Celluclast + GH10 Xylanase A + 35 10.57% GH62 Arabinofuranosidase ACelluclast + GH10 Xylanase A + 35 10.73% GH62 Arabinofuranosidase B

As shown in table 1 the addition of GH62 Arabinofuranosidase A and GH62Arabinofuranosidase B on top of Celluclast+GH10 Xylanase A cansignificantly increase the yield of starch+gluten in corn wet-millingprocess.

Example 9: Use of GH10 Xylanase and/or GH62 Arabinofuranosidase

10-g fiber assay is performed at pH 3.8, incubating at 52° C. for 1 hourat dose of 30 ug enzyme protein per gram corn, using a blend includingCelluclast and GH10 Xylanase A, in combination with either GH62Arabinofuranosidase A or GH62 Arabinofuranosidase B. Blend consists of5% GH62 Arabinofuranosidase A or GH62 Arabinofuranosidase B, 15% of GH10Xylanase A, and the remaining 80% from Celluclast. For comparison, blendcontaining Celluclast and GH10 Xylanase A only (no GH62) was included.The corn fiber with 16.67% residual starch and 10.77% residual proteinwas used as substrate in the fiber assay. Release of starch+gluten (drysubstance) as well as individual starch and protein from corn fiber atthe specified doses below was measured.

TABLE 2 Dose (ug enzyme Starch + Individual Individual protein/g GlutenStarch Protein Treatments corn) Recovered Recovered Recovered No Enzyme0  9.75% 4.95% 4.80% Celluclast + GH10 30 13.95% 8.29% 5.66% Xylanase ACelluclast + GH10 28.5 13.50% 8.03% 5.47% Xylanase A Celluclast + GH1030 15.40% 9.58% 5.82% Xylanase A + GH62 Arabinofuranosidase ACelluclast + GH10 30 15.20% 9.46% 5.74% Xylanase A + GH62Arabinofuranosidase B

As shown in table 2 the addition of GH62 Arabinofuranosidase A and GH62Arabinofuranosidase B on top of Celluclast+GH10 Xylanase A cansignificantly increase the yield of starch+gluten in corn wet-millingprocess.

Example 10: Use of GH10 Xylanase, GH30 Xylanase and/or GH62Arabinofuranosidase

A 10-g fiber assay was performed at pH 3.8, with incubation at 52° C.for 1 hour and a dosage of 35 ug enzyme protein per gram corn, usingenzyme blends containing GH10 Xylanase A, GH30 Xylanase A, GH62Arabinofuranosidase A, and Celluclast with the detailed ratio of 35 ugEP/g corn as below table.

TABLE 3 GH10 GH30 GH62 Xylanase Xylanase Arabinofur- Celluclast A Aanosidase A (ug-EP/g (ug-EP/g (ug-EP/g (ug-EP/g Treatments corn) corn)corn) corn) No Enzyme 0 0 0 0 Celluclast 35 0 0 0 Celluclast + GH10 285.25 0 1.75 Xylanase A + GH62 Arabinofuranosidase A Celluclast + GH10 285.25 1.75 0 Xylanase A + GH30 Xylanase A Celluclast + GH30 28 0 5.251.75 Xylanase A + GH62 Arabinofuranosidase A Celluclast + GH10 28 3.51.75 1.75 Xylanase A + GH30 Xylanase A + GH62 Arabinofuranosidase A

For comparison, an enzyme composition containing only Celluclast wasincluded. A corn fiber with 15.52% residual starch and 12.00% residualprotein in fiber was used as substrate in the fiber assay. Release ofstarch+gluten (dry substance) from the corn fiber at the specifieddosage was measured; the results are provided in the table 4 below.

TABLE 4 Dose Starch + (ug enzyme Gluten Treatments protein/g corn)Recovered No Enzyme 0 4.39% Celluclast 35 6.68% Celluclast + GH10Xylanase A + GH62 35 9.40% Arabinofuranosidase A Celluclast + GH10Xylanase A + GH30 35 9.45% Xylanase A Celluclast + GH30 Xylanase A +GH62 35 8.55% Arabinofuranosidase A Celluclast + GH10 Xylanase A + GH3035 9.70% Xylanase A + GH62 Arabinofuranosidase A

As shown in table 4, the addition of combined GH62 ArabinofuranosidaseA+GH30 Xylanase A on top of Celluclast+GH10 Xylanase A can significantlyincrease the yield of starch+gluten in corn wet-milling process.

Example 11: Preparation of Sterilized Palm Fruit Mesocarp

Step Action 1 In palm oil mill, oil palm FFBs (fresh fruit bunches) arereceived directly from the field, subjected to sterilization inindustrial autoclave (120° C. for 120 minutes) and then threshed toobtain oil palm fruitlets along with calyx leaves. Collect thesterilized palm fruitlets. 2 Separate the oil palm fruitlets and discardthe rest of the biomass [calyx leaves and small pieces of fruit bunchstalk (called as empty fruit bunch or EFB)]. 3 Pack the fruitlets in anautoclavable plastic cover and cook it in a kitchen pressure cooker (10L Capacity; Aluminum) for below induction cooktop program: Program Name:Pressure Cooker Time: 30 minutes Watt/Temperature: 1300 W/180° C. 4Spread the cooked fruitlets in a tray and allow it to cool down toapproximately 50° C. 5 Peel off the mesocarp from the nut. Collect themesocarp in a pre-weighed plastic storage container and record theweight. 6 Record the weight of nuts and discard it. 7 The peeledmesocarp is stored at 4° C. until use.

Example 12: Preparation of Substrate

The sterilized palm fruit mesocarp is pressed:

Step Action 1 Mash required amount of mesocarp in mash bath at ~200r.p.m. for: a. 3 minutes, if mesocarp quantity is more than 2 Kilograms.b. 2 minutes, if mesocarp quantity is 2-1 Kilograms. 2 Manually mix themashed mesocarp until it is uniformly mixed

Example 13: 10 gms Assay Protocol for Palm Substrate

1.10 g of prepared mash is aliquoted into 50 ml Falcon tubes withintermittent mixing to ensure substrate homogeneity. Note down the exactweight of substrate weighed;

2. Also, note down the empty weight of plastic pertriplates that are tobe used for collecting extracted oil;

3. Pre-condition the tubes with substrate, keeping them at 90° C. for 5minutes;

4. Transfer the tubes to respective incubation temperature (55° C.)water bath and pre-condition them for 10 minutes;

5. Inoculate the tubes with 500 μL of water in case of Control and 500μL of enzyme solution in case of other enzyme treatments;

6. After adding enzyme/water, mix the contents with a microspatula 5times in clock-wise and 5 times in anti-clockwise direction to ensureproper mixing;

7. Incubate for specified time (15 mins/30 mins) with intermittentmixing at every 15th minute of incubation with spatula, as specified inStep 6;

8. At the end of incubation, add 20 ml of water into each tube and mixwell;

9. For clarification, transfer the tubes to 90° C. water bath and allowit to clarify for 30 mins;

10. Centrifuge the tubes in table top centrifuge at 7000 rpm, 30° C. for10 min to get oil layer at the top;

11. Pipette out the oil layer into pre-weighed petriplates. Use hotwater to completely extract free oil from each tube;

12. Note down the weight of petriplates with extracted oil;

13. The oil yield can by calculated by: Oil yield=Weight of petri dishcontaining oil extracted—Weight of empty petri dish.

TABLE 5 mg Enzyme for Oil yield in Samples 10 g substrate grams Controlno enzyme 2.88 ± 0.2 GH10 Xylanase A + GH62 0.12 + 0.04 3.18 ± 0.1Arabinofuranosidase A

As shown in table 5 the addition of GH10 Xylanase A and GH62Arabinofuranosidase A can increase the oil yield from 10 gms of palmmesocarp.

What is claimed is:
 1. A process for treating crop kernels, comprisingthe steps of: a) soaking kernels in water to produce soaked kernels; b)grinding the soaked kernels to form ground kernels; c) separating thegerm from the ground kernels to produce a slurry comprising fiber,starch and protein; and d) treating the slurry in a fiber washing stepto separate the fiber from the starch and protein in the presence of aneffective amount of a polypeptide having GH62 arabinofuranosidaseactivity and a polypeptide having GH10 xylanase activity, wherein thepolypeptide having GH62 arabinofuranosidase activity has at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 1 and thepolypeptide having GH10 xylanase activity has at least 95% sequenceidentity to the mature polypeptide of SEQ ID NO:
 4. 2. The process ofclaim 1, which further comprises a starch gluten separation step andstarch washing step.
 3. The process of claim 1, wherein the soaking isperformed in the presence of between 0.01-1% SO₂ and/or NaHSO₃.
 4. Theprocess of claim 1, wherein the crop kernels are from corn (maize),rice, barley, sorghum bean, fruit hulls, or wheat.
 5. The process ofclaim 1, further comprising treating the soaked crop kernels or afraction of said crop kernels in the presence of one or morecellulolytic enzymes.
 6. The process of claim 1, wherein said cropkernels or a fraction of said crop kernels is admixed with said one ormore hydrolytic enzymes.
 7. The process of claim 1, further comprisingtreating the soaked crop kernels or a fraction of said crop kernels inthe presence of a polypeptide having GH30 xylanase activity.
 8. Theprocess of claim 1, further comprising treating the soaked crop kernelsor a fraction of said crop kernels in the presence of an enzyme selectedfrom the group consisting of a cellulolytic enzyme or a cellulase, anendoglucanase, a protease, a cellobiohydrolase I, a cellobiohydrolaseII, a GH61 polypeptide, or a combination thereof.
 9. The process ofclaim 1, wherein the fiber washing step comprises a space configured toprovide a total retention time in the fiber washing system of at least0.5 hour.
 10. The process of claim 1, wherein the polypeptide havingGH62 arabinofuranosidase activity has at least 97% sequence identity tothe mature polypeptide of SEQ ID NO: 1 and the polypeptide having GH10xylanase activity has at least 97% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 11. The process of claim 1, wherein thepolypeptide having GH62 arabinofuranosidase activity has at least 98%sequence identity to the mature polypeptide of SEQ ID NO: 1 and thepolypeptide having GH10 xylanase activity has at least 98% sequenceidentity to the mature polypeptide of SEQ ID NO:
 4. 12. The process ofclaim 1, wherein the polypeptide having GH62 arabinofuranosidaseactivity has at least 99% sequence identity to the mature polypeptide ofSEQ ID NO: 1 and the polypeptide having GH10 xylanase activity has atleast 99% sequence identity to the mature polypeptide of SEQ ID NO: 4.13. The process of claim 1, wherein the polypeptide having GH62arabinofuranosidase activity is the mature polypeptide of SEQ ID NO: 1and the polypeptide having GH10 xylanase activity is the maturepolypeptide of SEQ ID NO: 4.